Treatment using dantrolene

ABSTRACT

Provided are low-volume, safe for injection formulations of dantrolene yielding significant advantages over the currently approved and marketed dantrolene for malignant hyperthermia (MH) threatening anesthetic crisis. Once dantrolene can be made immediately available to patients triggered of MH, the anesthesiologist will be able to focus exclusively on the management of the patient&#39;s physiologic status in this complex and evolving crisis, not on the laborious and time consuming reconstitution process of the rescue agent. The low volume, safe for injection formulations of dantrolene have significant advantages over currently used approaches to the prevention and treatment of pumphead, and other neurological, cognitive and motor dysfunction incident to iatrogenically or trauma induced situations of altered blood flow, including those incurred during surgical procedures involving CPB or related procedures, as well as those incurred during non-normothermic episodes caused iatrogenically or by disease.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/353,478, filed Jan. 19, 2012, which is a continuation of U.S.application Ser. No. 12/717,588 filed Mar. 4, 2010 (now U.S. Pat. No.7,758,890), which is a continuation of U.S. application Ser. No.10/788,413 filed Mar. 1, 2004, which claims the benefit of U.S.Provisional Application Ser. Nos. 60/539,324 filed Jan. 28, 2004 and60/451,249 filed Mar. 4, 2003, and which is a continuation-in-part ofU.S. application Ser. No. 10/170,236 filed Jun. 13, 2002 (abandoned),which claims the benefit of U.S. Provisional Application Ser. No.60/300,482 filed Jun. 23, 2001, each of which is incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the prophylactic and therapeutic use inmammals, particularly man, of dantrolene and its salts, relatives andanalogs. Low volume safe for injection formulations of dantroleneprovide improved prevention and treatment for currently recognizedindications, including malignant hyperthermia, and enable practical useof dantrolene in the field, thus extending its pharmaceutical use tonovel applications. The invention further relates to the use ofdantrolene in the prophylaxis and treatment of cerebrospinal injury orand cognitive dysfunction secondary to iatrogenically induced states ofaltered blood flow, including those incurred during surgical proceduresinvolving CPB or related procedures and those which are trauma induced,including pumphead, as well as those incurred during non-normothermicepisodes caused iatrogenically or by disease.

DEFINITIONS

“Altered blood flow”—blood flow that exists, and thus has a nonzero flowrate, but is significantly different from normal. For altered blood flowthat represents a reduction in pressure, this is considered to begreater than a 10% decrease from baseline systolic pressure, orassociated decrease in mean arterial pressure, but less than 95%decrease. Pulsatile changes or temporary elevations in blood pressureare also to be considered altered blood flow.

“Central Nervous System (CNS)”—that portion of the nervous systemconsisting of the brain and spinal cord (pars centralis systematisnervosa (NA) and systema nervosum centrale (NA alternative)” (Dorland'sMedical Dictionary, p. 1652).

“Cerebrospinal System”—that portion of the nervous system comprised ofthe brain (cerebrum, cerebellum, and brainstem) and spinal cord (whiteand gray matter) to the level of the conus medularis, but absent thecranial nerves (CN I-XII) as well as the components of the peripheralnervous system.

“Colloidal”—in the current context, a formulation is colloidal if theactive compound is present in distinct particles which are primarilymicron or submicron in size, in particular less than about 100 micronsin average diameter, and in the present context more preferably lessthan about 2 microns in average diameter.

“Hypoxia”—a state of decreased oxygen supplies available to tissuesbelow normal physiologic levels despite adequate tissue perfusion thatcan induce states of neuropsychiatric changes and cognitive dysfunction.This may be induced by anemic hypoxia, histotoxic hypoxia, or stagnanthypoxia. Conditions of ventilation/perfusion mismatch as induced

by certain pulmonary disease conditions, mechanical or assistedventilation, or an inadequate concentration of oxygen (insufficientFiO2), may induce a state of hypoxia. Accidental hypothermia, such asthat associated with exposure, may also induce hypoxia.

“Low-mannitol” formulation means a formulation of dantrolene (or a saltthereof) that comprises less than 30 milligrams of mannitol permilligram of dantrolene.

“Low-volume formulation” means a formulation of dantrolene (or a saltthereof) that requires less than 100 ml of liquid, and preferably lessthan 10 ml of liquid, in order to deliver a therapeutic dose of about300 mg.

“Neuropathy”—a general term denoting functional disturbances and/orpathological changes in the peripheral nervous system.” (Dorland'sMedical Dictionary, p. 1652).

“Normothermia”—the preferred body temperature at which humans and mostmammals exist and thrive, normally a very narrow temperature range (theinterthreshold range), being auto-regulated chiefly by the hypothalamus.Hypothermia in humans is largely regarded as being a core bodytemperature of less than 36 degrees C. In humans, raising thetemperature even a fraction of a degree induces vasodilatation andsweating, resulting in hyperthermia. While under the influence ofgeneral anesthesia, humans, and most mammals are considered to bepoikilothermic; that is, they lose the ability to reliably regulate astate of normothermia and their core body temperatures tend to drifttoward the ambient environmental temperature.

“Peripheral Nervous System”—that portion of the nervous systemconsisting of the nerves and ganglia outside the brain and spinal chord(pars peripherica systematis nervous (NA) and systema nervosumperiphericum (NA alternative).” (Dorland's Medical Dictionary, p. 1656).

“Safe for injection”. We define “safe for injection” to mean aformulation that can be reliably injected intravenously into appropriatetest subjects or model mammals, at relevant clinical doses, with a lowincidence of life-threatening complications due to the formulation,where low incidence means less than about 10% of cases, and preferablyless than about 1% of cases. In particular, formulation-relatedtoxicities, such as pulmonary emboli (PE) due to supermicron-sizedparticles or aggregates, pathologically altered arterial pressures, orsevere vascular damage, must be limited to low incidence. It isimportant to point out that in the context of the current patent, theterm “safe for injection” does not in any way imply a restriction of adrug formulation to intravenous injection, it merely means that theformulation is sufficiently safe so as to allow intravenous injection.The reason for focusing on the intravenous route with regard to thesafety issue is that even when a formulation is administered by anotherroute of injection, such as intramuscular, intra-arterial, subcutaneous,intraperitoneal, intraocular, or by local instillation, the danger ofinadvertent routing to a vein cannot be ignored, and often demands thatthe formulation be safe even if errant administration results in what isessentially an intravenous administration. Because of this, in thispatent we use the terms “safe for intravenous injection” and “safe forinjection” interchangeably.

“Salt of dantrolene”—a pharmaceutically acceptable salt of dantrolene,in which the counter ion to the dantrolene anion is chosen from thegroup consisting of sodium (the preferred counter ion), potassium,ammonium, calcium, or magnesium; other possible cations that could beused against dantrolene in the context of this invention includebenzyltrimethylammonium, tetramethylammonium, N-methylpyridinium,tetrabutylammonium, 2-(2,3-dihydroxy-1-propylamino)-quinolizinium,Safranine O,

quinolizinium, quinolizinium, 2-carbamoyl-1-methylpyridinium,2,3-dimethyl-1-phenyl-4-trimethyl-ammonium-3-pyrazolin-5-one,dimethylammonium, 1,3-dimethylimidazolium,2,3-dimethyl-1-phenyl-4-trimethyl-ammonium-3-pyrazolin-5-one,2-(1-hydroxy-2-methyl)propyltri-methylammonium, and choline.

“Treatment”, “Therapeusis”—each of these terms includes bothprophylactic (pretreatment) and therapeutic treatment.

DESCRIPTION OF THE PRIOR ART

Altered or impaired cognitive function, neuropsychiatric changes, andmotor function are associated with non-specific mechanisms linked todecreased systemic blood pressure, decreased cerebral perfusion andperfusion pressures, and low blood flow states.

Complete interruption of blood supply and embolic phenomena associatedwith the localized cessation of blood flow, as in the case of stroke,ischemia, and resultant reperfusion injuries are known to initiate acomplex cascade of physiologic events, causing peripheral damage, aswell defined by Mangat et al. in U.S. Pat. No. 6,462,066. Localizedblood flow cessation and subsequent reperfusion in the peripheralvessels of the eye, and associated visual disorders, were of particularfocus in the latter patent. The neuropathies that were the subject ofthe patent by Mangat et al. are all in fact peripheral by definition,according to the standard definition of the term “neuropathy” asrecorded by, for example, Dorland's Medical Dictionary (p. 1652).

However, various iatrogenically induced events as well as trauma mayalter systemic blood pressure in a much less dramatic fashion, e.g.,temporarily decreasing cerebral blood flow, and it is now beginning tobe recognized that such altered blood flow, manifested in the centralnervous system, can induce neuropsychiatric changes, impair cognitivefunction, and alter motor function and control. Such alterations mayresult in either self-limiting or permanent neurologic sequellae. Suchconditions are not recognized in the patent of Mangat et al. as beingtreatable by the methods of that patent, and in fact are not the subjectof specific medication-based preventive measures in the current medicalpractice. Nor are cerebrospinal conditions resulting from such alteredblood flow situations recognized as preventable in the US PatentApplication Pub. No. 2004/0006124 of Dong, which focuses onneuroretinopathies.

In the case of CNS disturbances, administration of potentiallytherapeutic agents by injection can be highly problematic, evendangerous, when standard compositions and protocols that apply intreatment of peripheral disturbances are applied in accordance withordinary skill in that art. In particular, for the treatment of CNSdisturbances, large volumes of administration and the presence ofexcipients that compromise the blood-brain barrier are oftencontraindicated. Such complications are neither recognized nor addressedby U.S. Pat. No. 6,462,066, further underscoring the limitation of thatsubject matter to peripheral tissues.

A number of therapeutic agents have been discussed or experimented within attempts to prevent or reduce cerebrospinal damage resulting fromischemic stroke. These include DP-b99, nimodipine, flunarizine, ebselen,tirilazad, clomethiazole, diazepam, GYKI 52466, NBQX, YM90K, YM872,ZK-200775, SYM 2081, AR-R15896, aptiganel, dextromethorphan, magnesium,memantine, MK-801, NPS 1506, remacemide, ACEA 1021, GVI50526, eliprodil,ifenprodil, FGF, Anti-ICAM, Hu23F2G, lubeluzole, naloxone, nalmefene,citicoline, Bay x 3072 repinotan, fosphenyloin, 619C89, BMS-204352,cerebrolysin, and piracetam. Most if not all of these attempts haveresulted in little if any improvement. See Sandercock et al., HealthTechnology Assessment, 2002, vol. 6(26), page 27.

U.S. Pat. No. 6,187,756 to Lee focuses on treatment of disordersmediated by Amyloid Precursor Protein (APP), such as Alzheimer'sdisease, in particular on the use of beta-adrenergic receptorantagonists. U.S. Pat. No. 5,506,231 to Lipton deals with disordersmediated by the HIV-1 coat protein gp120. While these patents deal withCNS disorders, they do not teach of treatments, nor especiallypre-treatments, for disorders that result immediately—including inhumans not previously suffering from factors threatening cerebrospinalhealth—from altered blood flow such as that associated withcardiopulmonary bypass and other surgical procedures.

Dantrolene is the rescue agent of choice in the treatment of malignanthyperthermia and is therefore widely available in most locations whereanesthetics are delivered. First synthesized in 1967, dantrolene wasused initially in the treatment of muscle spasms in 1975, and laterreceived FDA approval in 1979 for treating the crisis of MH. Morebroadly, dantrolene is of value in a range of other conditions requiringa powerful muscle relaxant and treatment against nerve spasticity. Asparticularly important examples, dantrolene has been of interest and usein the prophylaxis and treatment of other life-threatening conditionssuch as overdose from recreational drugs such as “ecstasy”(N-methyl-3,4-methylene-dioxyphenylisopropylamine, CAS #42542-10-9),heat stroke, neuroleptic malignant syndrome, and ischemic damage to theperipheral nervous system, and may be of importance in the prevention ofsudden infant death syndrome (SIDS).

A derivative of hydantoin-furan, dantrolene sodium is poorly soluble inwater. The currently marketed formulation, Dantrium® Intravenous,(Proctor & Gamble, Cinn, Ohio) exists in a lyophilized state, containing20 mg of dantrolene sodium and 3000 mg of mannitol in a 70 ml sterilevial. A final concentration of 0.33 mg/ml of dantrolene and 50 mg/mlmannitol is achieved upon reconstitution with 60 ml sterile water. Assuch, this formulation exhibits a number of undesirable properties duein large part to the poor solubility characteristics of dantrolene.These problems have been well described by others and include cumbersomeand some times imprecise preparation, significant time and elevatedtemperatures to prepare a solution suitable for intravenousadministration (Grass et al), large volume of solution (600 ml minimumfor individual) to deliver an efficacious dose typically ranging from2.5 to 10.0 mg/kg. See MHAUS, H. Rosenberg, Clinical Anesthesia, 4th Ed.The pH of 9.5 in current formulations is irritating and increases thepotential for tissue necrosis secondary to extravasation and endothelialvascular damage (thrombophlebitis). Dissolution of the currentlymarketed Dantrium® formulation according to the protocol currentlypracticed in actual MH crises has been shown to be incomplete, stronglyindicating that large crystals of dantrolene are being injectedintravenously in patients whose cardiovascular state is already underextreme stress. Furthermore, the large loading of mannitol in currentlymarketed formulations can cause CNS complications.

Deaths have been attributed to the cumbersome preparation of thecurrently marketed dantrolene formulation, due to the excessive time andeffort required for reconstitution, as well as the resultant inaccuratedosing and lack of portability.

Current recommendations by the Malignant Hyperthermia Association of theUnited States (MHAUS) stipulate that all locations where generalanesthetics are administered have 36 vials of dantrolene sodium (720 mg)on hand at all times. In most operative suites, a dedicated MH Cart isequipped with 6 six packs of Dantrium® and liters of sterile water forits reconstitution, as well as 60 cc syringes and needles with which toprepare it, and central line kits, sodium bicarbonate, and otherdisposables required for administration via central access as opposed toperipheral veins. Reconstitution and injection of several dozen vials ofDantrium® in the face of a fulminant MH crisis is a daunting task, oftenrequiring the assistance of several anesthesia personnel, and these timeconstraints frequently result in incomplete dissolution of thedantrolene prior to injection and/or treatment delays that can result inharm to the patient.

Others have previously reported their experiences in attempting toimprove upon the currently available product. Phospholipid-coatedmicrocrystal formulations (see, viz., U.S. Pat. No. 5,091,188) ofdantrolene and dantrolene sodium have been evaluated in normal and MHSswine, but found to be unsafe for injection. See Karan et al., Anesth.Analg. 1996, 52:796. Investigators were challenged in creating uniformparticle sizes, ranging in size from 500 nm to 6 microns in size, whichresulted in variable treatment results and cardiovascular collapse insome swine believed to be due to either large particles or spontaneousparticle agglomeration resulting in pulmonary emboli.Phospholipid-coated dantrolene sodium was found to aggregate, making itunacceptable for injection, and formulations of the free acid form(dantrolene) failed in a significant fraction of cases tested, includingwhen used as a pretreatment prior to exposure to halothane andsuccinylcholine in pigs (a standard, accepted test for dantroleneeffectiveness). Incidences of death and severe complications due to theformulation in these studies was significantly greater than 10% of theanimals tested. Furthermore, the phospholipid-coated dantrolene crystalformulation was significantly less potent than the marketed Dantrium®formulation in twitch tension tests on rats, with the reported ED50being 1.0 mg/kg instead of the 0.6 mg/kg for the marketed formulation,and there is reason to believe that pharmacokinetics may have beensignificantly retarded as well.

Dantrolene sodium in solution over time precipitates the free acid form,which is unacceptable for an injectable formulation. This probablyprecludes the possibility of an aqueous formulation of dantrolene sodiumwith adequate shelf-life. Nevertheless, for a dry formulation, the finaladministration of the formulation will generally involve reconstitutioninto an injectable liquid, which is typically, and preferably, water.

Methods have been described in the literature for the preparation ofcolloidal suspensions of pharmaceutical compounds, including those thatare pharmaceutically-acceptable for injection. Usually the crystals arefreeze-dried into a powder that can be re-dispersed in water. Thisapproach has mainly been used for orally administered formulations. Forinjectable formulations, it is crucial that the powder re-disperse as anultrafine dispersion, with an extremely low incidence of particles oraggregates greater than 1 micron in effective size. In those unusualinstances where attempts have been made to prepare dried powderformulations for rapid reconstitution into a safe-for-injectiondispersion, some attempts have failed due to the unsuitability of theactive drug compound for the methods used. This is particularly true forcompounds that have appreciable solubility in water, since mostpharmaceutical milling processes are based on aqueous milling.

Dantrolene sodium, the form of dantrolene currently marketed inDantrium® is currently designed to be reconstituted as an aqueoussolution (as opposed to dispersion) for injection, leading to the tacitassumption that its water solubility may be prohibitively high for thesestandard methods, and likewise to formulation efforts focused on the useof water-insoluble coatings. The latter (in particular thephosphatidylcholine-based coatings investigated by Karan et al.) haveproven unsafe for injection, and are in general contraindicated in thecase of dantrolene since rapid onset of action is imperative, andbecause water-insoluble coatings can increase toxicity on injection dueto particle size issues.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide dantrolene, or oneof its salts, analogs or relatives, in a pharmaceutically acceptableformulation that can deliver the requisite amount of drug in a liquidvolume that is greatly reduced from that required by the currentlymarketed injectable Dantrium® formulation (which requires volumes on theorder of 500 ml to 1800 ml for a human application), and which thereforeminimizes or circumvents the complications and dangers associated withreconstituting large liquid volumes of multiple vials of lyophilizedagent for administration, including but not limited to the treatment ofsome of the conditions of focus in this patent.

This substantial reduction in volume and associated problems is notforeseen in the Mangat et al. patent, but should be considered of highimportance in view of, for example, the added complications imposed when500-1,800 ml of aqueous solution must be reconstituted and administeredin a procedure whose success is dependent on rapid intervention,critical control of an extracorporeal circuit, and/or whereintravascular volume expansion may be relatively or absolutelycontraindicated. With certain embodiments of the current invention, adantrolene dose of up to 500 mg can be delivered in a liquid volume lessthan or equal to about 150 ml; a 300 mg dose can be delivered in avolume of less than or equal to about 100 ml, more preferably less thanor equal to about 30 ml, and most preferably less than or equal to about5 ml. The latter volume is sufficiently small that the entireformulation could be loaded into an auto injector in accordance withstandard volumes of such devices, thus providing for portability asrequired by field and ambulance applications.

It is a further object of this invention to provide a low-volumeformulation of dantrolene or one of its salts that is either a solution,or contains particles that are sufficiently small to permit safeadministration via all conceivable routes, certainly including but notlimited to intravenous, intramuscular, intrathecal, and extracorporealfluids and/or circuits, in particular such that over 95% of theparticles are less than 0.8 microns, or preferably less than 0.45microns. Such sizes are important not only for safety against pulmonaryemboli on injection, but also against microbial infections since theycan allow for filtration, e.g., using an in-line filter, at sizes thatexclude at least some of the most important microbes.

It is a further an object of this invention to provide formulations ofdantrolene that are rapidly and reliably reconstituted in emergencyclinical situations, as well as in non-emergency and prophylacticcircumstances. In particular, the formulations will be such that a fulltherapeutic dose of 300 mg can be reconstituted in a clinical situationin under 1 minute by a single clinician.

It is a further object of this invention to provide a dry powderformulation of sodium dantrolene, or of another salt of dantrolene, thatshows minimal appearance of the free acid dantrolene during storage.

It is a further object of this invention to provide a pharmaceuticallyacceptable low-volume dantrolene sodium formulation that contains lowmannitol content, less than about 30 mg of mannitol per milligram ofdantrolene, thus permitting safer use in indications where neurologicalcomplications may occur.

It is an object of this invention to provide a method for treatingmalignant hyperthermia and other and related conditions as identified inthis application, including but not limited to MDMA overdose and heatstroke, by the use of a safe-for-injection, low-volume colloidalsuspensions of dantrolene or one of its salts.

It is a further object of this invention to provide safe, low-volume andlow-mannitol colloidal suspensions of dantrolene that overcome theseproblems, improving treatment of MH and related conditions in theoperating room, and making efficacious treatment more widely availablein other settings, and other etiologies. Thus, it is an object of theinvention to provide dantrolene, or one of its salts, analogs orrelatives, in a pharmaceutically acceptable formulation that can deliverthe requisite amount of drug in a liquid volume that is greatly reducedfrom that required by the currently marketed injectable Dantrium®formulation (which requires volumes on the order of 500 ml to 1800 mlfor a human application), and which therefore minimizes or circumventsthe complications and dangers associated with reconstituting largeliquid volumes of multiple vials of lyophilized agent foradministration, including but not limited to the treatment of some ofthe conditions of focus in this patent.

Another aspect of this invention centers around a class of newindications for the use of the dantrolene. In particular, it is anobject of this invention to provide a method by which to prevent, reduceor reverse the negative cerebrospinal and cognitive injuries, describedherein, which can be associated with altered, and especially decreased,blood pressures; altered, and especially decreased, blood flow; altered,and especially decreased cerebral perfusion; altered, and especiallydiminished pulsatile flow, as well as increased intracranial pressureswhich inherently alter, and especially impair cerebral perfusion andsubsequent oxygenation of cerebral tissues; and non-normothermic statesespecially those which are sustained for more than about four hours. Thephenomena of altered cognitive abilities and function as well asneuropsychiatric changes with or without impaired motor function iscommonly referred to as “pumphead” among anesthesiologists,cardiothoracic surgeons, and certain other medical personnel. Inparticular, in this patent, it is envisioned that the prophylacticadministration of dantrolene, or one of its salts, analogs or relatives,may prevent or limit the effects of these neurological complications viaa unique and synergistic combination of a number of intracellular and/ormetabolic mechanisms, and via stabilization of intracellular calcium. Itis further expected that dantrolene will be a suitable treatment agentcapable of minimizing neurological complications when provided in amanner timely to the insult, not only in humans but potentially inveterinary settings as well.

DETAILED DESCRIPTION OF THE INVENTION

The current invention focuses on new formulations, and indications, ofdantrolene and dantrolene salts that are safe for injection and requireonly small liquid volumes for administration, less than about 100 ml andpreferably less than about 10 ml for the administration of a typicaltherapeutic dose of about 300 mg. It is largely anticipated that thisinvention will allow for unit dosing in convenient, single-doselyophilized or predispersed material. This will allow for accurateadministration either corporally or extracorporally with a minimum ofmanipulation. The large-volume workup of the current Dantrium®formulation greatly interferes with the practicality of field use ofdantrolene, such as in military or ambulance applications, whereas thelow-volume formulations presented herein could be especially useful insuch field applications. Similarly, the invention could have value inpublic health situations requiring administration away from the clinic,such as in the event of a disease epidemic, or wartime orterrorist-related injuries, etc.

These formulations are colloidal suspensions of dantrolene or its saltsin a pharmaceutically acceptable liquid, preferably chosen from thegroup consisting of water, glycerol, propylene glycol,dimethylacetamide, ethanol, polyethylene glycol (e.g., PEG 300, PEG 400,PEG 3350), triethyl citrate, triacetin, monothioglycerol, or mixturesthereof, more preferably water or a water-miscible solvent, and mostpreferably water. The invention also discloses dry powder formulationsof dantrolene or one of its salts that can be rapidly (less than oneminute) reconstituted by adding a pharmaceutically acceptable liquid,preferably sterile water for injection, and mechanically agitating,preferably by hand shaking.

Another significant advantage of the invention as described herein, inaddition to providing safe-for-injection, rapidly reconstitutable andadministrable dantrolene formulations, is the reduction or omission ofmannitol from the currently marketed formulation. Mannitol functions asan intravascular osmotic gradient inducer drawing extravascular fluidsto the intravascular space. This may prove beneficial in treatingcertain types of edemas. However, in many surgical situations involvingneurological complications, mannitol is widely recognized to becontraindicated. In such a state, the mannitol leaves the intravascularspace, becoming extravascular and collecting in the region of thedisrupted blood brain barrier. Extravascularly, it creates a similarosmotic gradient, but here it causes free fluid accumulation in thecerebral tissue, increasing cerebral edema, increasing intracranialpressures while decreasing cerebral blood flow via alteration ofcerebral perfusion pressures.

Furthermore, an additional advantage of the precisely controllednanoparticle size of our colloidal suspension is that distribution ofdantrolene to poorly perfused skeletal muscle in a state of activetetany can be maximized. It has been theorized that in some instances offailed treatment of MH, that the crisis had evolved to a point wheretetanic contraction of muscle severely interrupted the delivery oflarger sized particles or crystals of dantrolene, rendering itunavailable to the binding site while appropriate concentration wereachieved elsewhere in the intravascular compartment.

It is anticipated that a wide range of doses of this low-volume,low-mannitol dantrolene sodium formulation will obtain the intendedeffect of alleviating an MH crisis or related event. A lower volumeformulation as provided herein, will allow for easier and more accurateadministration in a more rapid manner than prior art formulations. Atthis time, it is expected that doses ranging from 0.1 to 10.0 mg/kg willprove efficacious, depending upon the age, pre-existing state of health,and possible extent of neurologic injury depending upon the type andextent of the insult. The preferred range is about 0.5 to about 4 mg/kg.

In addition, another aspect of the invention is the discovery of newindications for dantrolene, for which existing dantrolene formulationsas well as low-volume formulations as disclosed herein provide for a newmethod of treatment and prophylaxis. The inventors have recognized thatdantrolene provides a surprising and synergistic combination ofbiochemical and pharmacologic mechanisms that make it of uniqueapplicability in the prevention and treatment of certain cerebrospinal,and especially cognitive, injuries which prior to this invention werepoorly understood and even more poorly treated. Attention to suchinjuries, particularly when their symptomology is “silent”, andsometimes delayed, following in the aftermath of certain surgicalprocedures, has in previous medical practice taken a back seat to theprimary surgical indication. Of these injuries, cognitive loss sometimesreferred to as “pumphead” is a representative example.

Materials and Methods for Making Colloidal Dantrolene of the CurrentInvention

Colloidal dispersions of submicron crystals of dantrolene or one of itssalts, that are safe for injection, can be prepared according to knownmethods of particle size reduction in pharmaceutical patents,literature, and practice. High-pressure homogenization and wet-millingare two general methods. For a representative discussion of millingtechniques in pharmaceutical settings see, for example, U.S. Pat. No.5,858,410 (which is herein incorporated by reference). An importantaspect of this invention is the realization that the water solubility ofsodium dantrolene is low enough that these methods can in fact beapplied. This is of fundamental importance because not only is thesodium salt of dantrolene the salt that is in the currently marketedformulation of dantrolene, with a long history of safe use, but also ourwork indicates that the dissolution of the free acid, for example, issignificantly slower and more problematic than that of dantrolenesodium, which is of importance in the safety of an injectable product.The Examples below illustrate these two general methods of production.Other methods include dry-milling, chemical precipitation, spray-drying(e.g., from aqueous solution, generally containing a stabilizer asdiscussed below), sonication, solvent-removal from template emulsions,evaporative precipitation into aqueous solutions, and supercriticalfluid-based methods such as Precipitation with Compressed Antisolvent.

Alternatively, more complicated microparticles can be produced whichcontain dantrolene or one of its salts dispersed or dissolved within thecore of the microparticle. For example, submicron dantrolene crystalscan be embedded within lyotropic liquid crystals, which in turn can becoated, as per U.S. Pat. No. 6,482,517 (which is herein incorporated byreference), or within particles or microfibers of one or morebiocompatible polymers, such as PLGA, collagen, carboxymethylcelluloseor other cellulosic polymer, albumin, casein, PVP, etc.

The size of the particles of dantrolene or dantrolene salt or relativein the formulation as per this invention is very important, particularlyin determining whether it is safe for injection. It should also be notedthat in the case of a lyophilized, dry powder formulation as per thisinvention, particles of drug (dantrolene or one of its salts, relatives,or analogs) which are present in the dry formulation in submicronparticle sizes may nevertheless be embedded in solids that are muchlarger, even as large as millimeters in size, provided that these lattersolids are readily dissolved in the carrier liquid (usually water) thatis added during reconstitution. For example, submicron crystals ofdantrolene sodium could be embedded in a solid or amorphous saccharide,such as lactose or trehalose, in which case the size of the overallsolid particles could be much larger than submicron; addition of waterwould quickly dissolve the saccharide in this case, and leave behindsubmicron crystals of drug, making the reconstituted formulation safefor intravenous injection.

In a dry powder formulation, in addition to sufficiently small(generally less than about 2 microns, and preferably less than about 0.8microns and more preferably less than about 0.45 microns) dantroleneparticle size, another feature that is important, and which alsodistinguishes dried formulations of this invention from prior art driedforms of dantrolene, is that the surface chemistry of the formulationensures dispersibility, upon reconstitution. In particular, theincorporation of stabilizers and in some cases dispersants (or,components such as PVP which can serve as both stabilizer anddispersant) in the dried formulation as discussed herein is done so asto ensure dispersibility upon addition of liquid, usually sterile waterfor injection. In contrast, addition of 3 to 150 milliliters of water toa simple powder of dantrolene sodium (as received from Sigma-AldrichChemical Company, for example), or to the Dantrium formulation, andsubsequent shaking by hand at room temperature would not yield a safefor injection dispersion, since the particle size would be too large, asreported by Mitchell and Leighton in Gen. Anesth., 2003, vol. 50(2), p.127. Furthermore, the absence of stabilizers in these cases would yieldparticles that would very quickly begin to settle into the bottom of areconstitution vial, or syringe.

The colloidal suspensions of dantrolene or its salts in the currentinvention comprise crystals of dantrolene, a dantrolene salt, or arelated muscle relaxant compound suspended or dispersed in apharmaceutically acceptable liquid, preferably chosen from the groupconsisting of water, glycerol, propylene glycol, dimethylacetamide,ethanol, polyethylene glycol (e.g., PEG 300, PEG 400, PEG 3350),triethyl citrate, triacetin, monothioglycerol, or mixtures thereof, morepreferably water or a water-miscible solvent, and most preferably water.Broadly, a stabilizer is usually required in order to achieve a stable,fine dispersion of crystals (or amorphous drug substance), and thestabilizer if required is preferably chosen in accordance with thefollowing. Stabilizers of use include select proteins, polymers, andsurfactants. The proteins of potential use as stabilizers includealbumin, casein, and salts of casein. Polymers includepolyvinylpyrrolidone (PVP), acacia (gum arabic), carmellose sodium,dextran, collagen, gelatin, gelatin hydrosylate, sodium starchglycolate, inulin, and xanthan. Suitable surfactants or block copolymercomponents (or mixtures thereof) may include:

a. cationic surfactant

b. anionic surfactant

c. semipolar surfactant

d. zwitterionic surfactant

-   -   i. in particular, a phospholipid    -   ii. a lipid mixture containing phospholipids, designed to match        the physico-chemical characteristics of a biomembrane

e. monoglyceride

f. PEGylated surfactant

g. one of the above but with aromatic ring

h. block copolymer

-   -   i. with both blocks hydrophobic, but mutually immiscible    -   ii. with both blocks hydrophilic, but mutually immiscible,    -   iii. with one block hydrophilic and the other hydrophobic, i.e.,        amphiphilic)

i. a mixture of two or more of the above.

Suitable lipids include phospholipids (such as phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, or sphingomyelin), orglycolipids (such as MGDG, diacylglucopyranosyl glycerols, and Lipid A).Other suitable lipids are phospholipids (including phosphatidylcholines,phosphatidylinositols, phosphatidylglycerols, phosphatidic acids,phosphatidylserines, phosphatidylethanolamines, etc.), sphingolipids(including sphingomyelins), glycolipids (such as galactolipids such asMGDG and DGDG, diacylglucopyranosyl glycerols, and Lipid A), salts ofcholic acids and related acids such as deoxycholic acid, glycocholicacid, taurocholic acid, etc., gentiobiosyls, isoprenoids, ceramides,plasmologens, cerebrosides (including sulphatides), gangliosides,cyclopentatriol lipids, dimethylaminopropane lipids, and lysolecithinsand other lysolipids which are derived from the above by removal of oneacyl chain.

Other suitable types of surfactants include anionic, cationic,zwittenionic, semipolar, PEGylated, amine oxide and aminolipids.Preferred surfactants are:

anionic—sodium oleate, sodium dodecyl sulfate, sodium diethylhexylsulfosuccinate, sodium dimethylhexyl sulfosuccinate, sodiumdi-2-ethylacetate, sodium 2-ethylhexyl sulfate, sodiumundecane-3-sulfate, sodium ethylphenylundecanoate, carboxylate soaps ofthe form ICn, where the chain length n is between 8 and 20 and I is amonovalent counterion such as lithium, sodium, potassium, rubidium,etc.;

cationic—dimethylammonium and trimethylammonium surfactants of chainlength from 8 to 20 and with chloride, bromide or sulfate counterion,myristyl-gammapicolinium chloride and relatives with alkyl chain lengthsfrom 8 to 18, benzalkonium benzoate, double-tailed quaternary ammoniumsurfactants with chain lengths between 8 and 18 carbons and bromide,chloride or sulfate counter ions;

nonionic PEGylated surfactants of the form CnEm where the alkane chainlength n is from 6 to 20 carbons and the average number of ethyleneoxide groups m is from 2 to 80, ethoxylated cholesterol;

zwitterionics and semipolars—N,N,N-trimethylaminodecanoimide, amineoxide surfactants with alkyl chain length from 8 to 18 carbons;dodecyldimethylammoniopropane-1-sulfate, dodecyldimethylammoniobutyrate,dodecyltrimethylene di(ammonium chloride); decylmethylsulfonediimine;dimethyleicosylammoniohexanoate and relatives of these zwitterionics andsemipolars with alkyl chain lengths from 8 to 20.

Preferred surfactants, including preservatives which are used assurfactants, which are FDA-approved as injectables include benzalkoniumchloride, sodium deoxycholate, myristyl-gamma-picolinium chloride,Poloxamer 188 (Pluronic F-68). Pluronic F-127, polyoxyl castor oil andrelated PEGylated castor oil derivatives such as Cremaphore EL, ArlatoneG, sorbitan monopalmitate, Pluronic 123, and sodium 2-ethylhexanoicacid. Other low-toxicity surfactants and lipids, which are of at leastrelatively low solubility in water, that are preferred for the presentinvention for products intended for a number of routes ofadministration, include: acetylated monoglycerides, aluminummonostearate, ascorbyl palmitate free acid and divalent salts, calciumstearoyl lactylate, ceteth-2, choleth, deoxycholic acid and divalentsalts, dimethyldioctadecylammonium bentonite, docusate calcium, glycerylstearate, stearamidoethyl diethylamine, ammoniated glycyrrhizin, lanolinnonionic derivatives, lauric myristic diethanolamide, magnesiumstearate, methyl gluceth-120 dioleate, monoglyceride citrate,octoxynol-1, oleth-2, oleth-5, peg vegetable oil, peglicol-5-oleate,pegoxol 7 stearate, poloxamer 331, polyglyceryl-10 tetralinoleate,polyoxyethylene fatty acid esters, polyoxyl castor oil, polyoxyldistearate, polyoxyl glyceryl stearate, polyoxyl lanolin, polyoxyl-8stearate, polyoxyl 150 distearate, polyoxyl 2 stearate, polyoxyl 35castor oil, polyoxyl 8 stearate, polyoxyl 60 castor oil, polyoxyl 75lanolin, polysorbate 85, sodium stearoyl lactylate, sorbitansesquioleate, sorbitan trioleate, stear-o-wet c, stear-o-wet m,stearalkonium chloride, stearamidoethyl diethylamine (vaginal),steareth-2, steareth-10, stearic acid, stearyl citrate, sodium stearylfumarate or divalent salt, trideceth 10, trilaneth-4 phosphate, DetainePB, JBR-99 rhamnolipid (from Jeneil Biosurfactant), glycocholic acid andits salts, taurochenodeoxycholic acid (particularly combined withvitamin E), tocopheryl dimethylaminoacetate hydrochloride, tocopherylphosphonate, tocopheryl peg 1000 succinate, cytofectin gs,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane, cholesterol linkedto lysinamide or ornithinamide, dimethyldioctadecyl ammonium bromide,1,2-dioleoyl-sn-3-ethylphosphocholine and other double-chained lipidswith a cationic charge carried by a phosphorus or arsenic atom,trimethyl aminoethane carbamoyl cholesterol iodide, lipoic acid,O,O′-ditetradecanoyl-N-(alpha-trimethyl ammonioacetyl)diethanolaminechloride (DC-6-14),N-[(1-(2,3-dioleyloxy)propyl)]-N—N—N-trimethylammonium chloride,N-methyl-4-(dioleyl)methylpyridinium chloride (saint-2), lipidicglycosides with amino alkyl pendent groups,1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide,bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide,N-hexadecyl-N-10-[O-(4-acetoxy)-phenylundecanoate]ethyl-dimethylammoniumbromide, bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide,3-beta-[N—(N′, N′-dimethylaminoethane)-carbamoyl]cholesterol, vaxfectin,cardiolipin, dodecyl-N,N-dimethylglycine, and lung surfactant (Exosurf,Survanta). Suitable block copolymers are those composed of two or moremutually immiscible blocks from the following classes of polymers:polydienes, polyallenes, polyacrylics and polymethacrylics (includingpolyacrylic acids, polymethacrylic acids, polyacrylates,polymethacrylates, polydisubstituted esters, polyacrylamides,polymethacrylamides, etc.), polyvinyl ethers, polyvinyl alcohols,polyacetals, polyvinyl ketones, polyvinylhalides, polyvinyl nitriles,polyvinyl esters, polystyrenes, polyphenylenes, polyoxides,polycarbonates, polyesters, polyanhydrides, polyurethanes,polysulfonates, polysiloxane, polysulfides, polysulfones, polyamides,polyhydrazides, polyureas, polycarbodiimides, polyphosphazenes,polysilanes, polysilazanes, polybenzoxazoles, polyoxadiazoles,polyoxadiazoiidines, polythiazoles, polybenzothiazoles,polypyromellitimides, polyquinoxalines, polybenzimidazoles,polypiperazines, cellulose derivatives, alginic acid and its salts,chitin, chitosan, glycogen, heparin, pectin, polyphosphorus nitrilechloride, polytri-n-butyl tin fluoride, polyphosphoryldimethylamide,poly.-2,5-selenienylene, poly-4-n-butylpyridinium bromide,poly-2-N-methylpyridinium iodide, polyallylammonium chloride, andpolysodium-sulfonate-trimethylene oxyethylene. Preferred polymer blocksare polyethylene oxide, polypropylene oxide, polybutadiene,polyisoprene, polychlorobutadiene, polyacetylene, polyacrylic acid andits salts, polymethacrylic acid and its salts, polyitaconic acid and itssalts, polymethylacrylate, polyethylacrylate, polybutylacrylate,polymethylmethacrylate, polypropylmethacrylate, poly-N-vinyl carbazole,polyacrylamide, polyisopropylacrylamide, polymethacrylamide,polyacrylonitrile, polyvinyl acetate, polyvinyl caprylate, polystyrene,poly-alpha-methylstyrene, polystyrene sulfonic acid and its salts,polybromostyrene, polybutyleneoxide, polyacrolein, polydimethylsiloxane,polyvinyl pyridine, polyvinyl pyrrolidone, polyoxy-tetramethylene,polydimethylfulvene, polymethylphenylsiloxane, polycyclopentadienylenevinylene, polyalkylthiophene, polyalkyl-p-phenylene,polyethylene-alt-propylene, polynorbornene,poly-5-((trimethylsiloxy)methyl)norbornene, polythiophenylene, heparin,pectin, chitin, chitosan, and alginic acid and its salts. Especiallypreferred block copolymers are polystyrene-b-butadiene,polystyrene-b-isoprene, polystyrene-b-styrenesulfonic acid,polyethyleneoxide-b-propyleneoxide, polystyrene-b-dimethylsiloxane,polyethyleneoxide-b-styrene,polynorborene-b-5-((trimethylsiloxy)methyl)norbornene,polyacetylene-b-5-((trimethylsiloxy)methyl)norbornene,polyacetylene-b-norbornene, polyethyleneoxide-b-norbornene,polybutyleneoxide-b-ethyleneoxide, polyethyleneoxide-b-siloxane, and thetriblock copolymer polyisoprene-b-styrene-b-2-vinylpyridine.

As discussed above, stabilizers that have significant water solubility,preferably greater than about 5 mg/ml, are inherently safer than thosewhich are less soluble than 5 mg/ml.

Methods for removing water from aqueous-based dispersions in order tocreate reconstitutable dry powders are well known to those skilled inthe art of parenteral products. Lyophilization, or freeze-drying, of anaqueous dispersion according to standard pharmaceutical procedures canbe applied to colloidal dispersions of dantrolene or one of its salts,preferably dantrolene sodium, so as to produce dry powders that can bereconstituted by the addition of sterile water for injection and shakingor vortexing. See for example U.S. Pat. No. 5,858,410. The use ofstabilizers that are solid at room temperature, as opposed to liquid,provides for a better freeze-dried product in general, and stronglyhygroscopic stabilizers are also less preferred. Preferred stabilizersfor the colloidal dispersions of the current invention thus includesodium deoxycholate, sodium dodecyl sulfate, PVP, benzalkonium chloride,sodium docusate, hydrolyzed gelatin, and the “F” Pluronics such as F-68and F-127. Albumin is to be avoided, particularly in large amountsrelative to the dantrolene, since albumin binds to dantrolene and thiscan interfere with the normal activity and pharmacokinetics of the drug.It should be noted that, as discussed elsewhere herein, highly insolublestabilizers are less preferred since they can interfere with thepharmacokinetics of dantrolene—unless, as illustrated in Example 4, theyare present (in the final, possibly reconstituted formulation) in theform of a nanoporous, reversed lyotropic liquid crystalline phase, suchas a cubic phase, which can actually promote absorption. Dispersingagents can also be added, such as saccharides like lactose, trehalose,sorbitol, sucrose, dextrose, mannitol, and such, with lactose, sorbitol,and mannitol especially preferred. Disintegrants, and particularlysuperdisintegrants, can be used to improve speed and efficiency ofreconstitution, and such compounds include PVP andcarboxymethylcellulose, both of which are safe for injection when usedin sufficiently low amounts.

The forms of dantrolene that can be used in the current inventioninclude dantrolene free acid and a range of pharmaceutically acceptablesalts of dantrolene, in which the counter ion to the dantrolene anion ischosen from the group consisting of sodium (the preferred counter ion),potassium, ammonium, calcium, or magnesium; other possible cations thatcould be used against dantrolene in the context of this inventioninclude benzyltrimethylammonium, tetramethylammonium,N-methylpyridinium, tetrabutylammonium,2-(2,3-dihydroxy-1-propylamino)-quinolizinium, Safranine O,

quinolizinium, quinolizinium, 2-carbamoyl-1-methylpyridinium,2,3-dimethyl-1-phenyl-4-trimethyl-ammonium-3-pyrazolin-5-one,dimethylammonium, 1,3-dimethylimidazolium,2,3-dimethyl-1-phenyl-4-trimethyl-ammonium-3-pyrazolin-5-one,2-(1-hydroxy-2-methyl)propyltri-methylammonium, and choline. Dantrolenefree acid can also be used, though it has been found in the course ofthis work that dissolution of formulations of the free acid are slowerand less reliable than in the case of a salt such as the sodium salt.The preferred salt of dantrolene is dantrolene sodium, the currentlymarketed salt.

The safety and greater portability and more appropriate package sizemade possible by the current invention will facilitate the broaderavailability of dantrolene to every surgical suite, emergency room, aswell as other specialty or surgical settings, as well as non-surgicaland non traditional settings wherever the need may arise, for treatmentof MH of any etiology, and for treatment of other indications. Suchindications that may be treatable by the colloidal dantroleneformulations of this invention include, but are not limited to, varioustypes of ischemia, heat stroke, overdose or reaction to recreationaldrugs such as “ecstasy”, neuroleptic malignant syndrome (NMS), centralcore disease (CCD), Duchenne Muscular Dystrophy (DMD), King-DenboroughSyndrome, Myoadenylate Deaminase Deficiency (MDD), Schwartz-Jampelsyndrome, the Fukuyama type of congenital muscular dystrophy,fibromyalgia, Becker muscular dystrophy, periodic paralysis, myotoniacongenita, sarcoplasmic reticulum adenosine triphosphatase deficiencysyndrome, Burkett's lymphoma, Sudden Infant Death Syndrome (SIDS),osteogenesis imperfecta, glycogen storage pathologies, mitochondrialmyopathies, and alterations in the endoplasmic reticulum associated withAlzheimer's disease, as well as toxic reactions to strychnine,phencyclidine, hemlock, amphetamines, MAO inhibitors, theophylline, LSDand other psychedelic drugs, and cocaine. In general the invention is ofpotential benefit in the treatment of seizures and musclecontraction-related hyperthermia, in conjunction with antipyretictreatment, as a muscle relaxant, and as a neuroprotective agent in theface of elevated cerebrospinal temperatures. The invention could also beof use in prophylactic treatment of MH during pregnancy. Broadlyspeaking, the invention can be applied in any condition where the lowvolume of administration is a significant advantage, including but notlimited to increasing portability, ease of use, reliability in dosing,timeliness of dosing, absence of larger undissolved solid material, andimproved safety in the face of neurological complications.

The colloidal dantrolene of this invention requires significantly lesstime for preparation and administration. At this time, we envisioncolloidal dantrolene will be made available as 3%-8% (30-80 mg/ml) in 5ml or 10 ml vials either as a stable suspension ready for injection oras a powder to be reconstituted in 10 ml or less of sterile water into asuspension ready for injection. It is anticipated that a fulltherapeutic dose could be delivered in less than one minute as a bolusinjection, easily attaining the 1 mg/kg/min, if not significantlyexceeding, recommended rate of administration. A reconstitutable powderwould be reconstituted by combining with sterile water for injection andshaken or vortexed; filtration prior to injection may be desirable.Reconstitutable powders of the invention can be reconstituted by asingle clinician in less than one minute to a safe-for-injectiondispersion.

The colloidal dantrolene of this invention may be formulated at a morephysiologic pH, likely reducing the risk of tissue damage and ofthrombophlebitis as associated with the extravasation of the currentDantrium® product at pH 9.5. This feature, coupled with the small bolusvolume of the colloidal product needed to be administered, will allowinjection via peripheral veins through small-bore cannulae (24 gauge),rather than the via central venous access as is frequently recommended.

Dantrolene is widely known to be a muscle relaxant. Therefore,protective measures may have to be undertaken, such as planning forendotracheal intubation and mechanical ventilation. While this techniqueis commonly practiced during general anesthesia for surgicalintervention and to facilitate hyperventilation in the management of thetrauma patient, there may be instances where it is impractical orcontraindicated to administer dantrolene given this concern. In theinstance of known adverse reaction by an individual to dantrolene, itsuse is contraindicated.

It is within the scope of this invention to provide a safe-for-injectiondispersion of dantrolene or one of its salts that is, or can be,pre-loaded into an autoinjector, particularly for field use. Aparticularly important application of such a formulation/device could bein military or terrorist arenas, where for example the use of chemicalor biological warfare agents may be a threat.

Other agents in place of, or in combination with, dantrolene and itssalts.

In place of, or in addition to, dantrolene salts, other agents mayprovide similar protection which may be useful as alternative colloidalformulations, or in conjunction with the colloidal dantrolenepreparations described herein. This is particularly true in cases wherethe agent has similar pharmacologic action as dantrolene sodium, andespecially if it is known to provide relief from MH. Thus, apharmacologically active relative of dantrolene, such as a compoundcontaining a hydantoin group and/or a nitrophenyl or nitrofuranyl group,which affects the ryanidine receptor and through it intracellularcalcium release, would be expected to be active within the presentinvention, particularly if it diminishes the symptoms of MH. As aninstructive example, while certain analogues such as azumolene arepharmacologically related to dantrolene and may be of use in the presentinvention, dantrolene would be preferred over azumolene because thelatter has shown limited benefit in the treatment of MalignantHyperthermia (MH); in contrast, dantrolene sodium is the mostefficacious rescue agent known for MH. It is also anticipated that newdantrolene analogs and chemical relatives will become available, and tothe extent that such a new agent has similar pharmacologic actions, andespecially to the extent that it relieves the symptoms of MH, it is tobe expected that the same agent can be used in the context of thepresent invention.

While dantrolene and its salts are the preferred agents, certain otheragents or classes of compounds, especially those agents known toregulate calcium intracellularly, could be of potential benefit insituations or conditions where the use of dantrolene sodium iscontraindicated. Calcium channel blockers as a class of drugs is oneexample of such a substitution. While the effects of such drugs oncalcium could be similar to those of dantrolene, it must be recognizedthat dantrolene has other pharmacologic effects that may be important inthe treatments of focus herein and may not be accomplished by such asubstitute.

EXAMPLES Example 1

Dantrolene sodium (synthesized by CarboMer, Inc.), in the amount of 2.40grams, was added to 27.60 gm of pH 10 buffer, into which had previouslybeen dissolved 0.24 gm of polyvinylpyrrolidone (PVP). This mixture wasthen loaded into a Model 110L Microfluidizer (Microfluidics Corp.,Bedford, Mass.), powered by a Kaeser air compressor. At a pump pressureof 15,000 psi, this was microfluidized for four cycles of 1.5 minuteseach.

At the end of this time, examination through a phase contrast opticalmicroscope with a 40× objective, and the particle size was seen to besubmicron for a high fraction of the drug crystals. A particle sizedistribution was then obtained using a Beckman Coulter N4Plus lightscattering particle sizer. The mean particle size was found to be 407nm, with a standard deviation of 21 nm, and 0.0% dust reported; togetherthese indicate an extremely well controlled particle size, and in a sizerange acceptable for intravenous injection. Zeta potential on theparticles was then measured with a Beckman Coulter DELSA 440SXinstrument, and indicated an average −54 mV potential and over 90% ofthe population lying between −80 and −25 mV. Such a strong zetapotential is sufficient to yield long-term dispersion stability viaionic stabilization.

For a dantrolene formulation made according to this protocol, a 240 mgdose of dantrolene sodium could be delivered in a volume ofapproximately 3 ml.

Example 2

Working in a nitrogen-filled glove box, dantrolene sodium, in the amountof 0.267 grams, was added to a 15 ml tube and then covered with 1.046 gmN,N-dimethylacetamide and 3.164 gm glycerol which had beenheat-sterilized. The dantrolene was dissolved in this mixture by acombination of stifling, vortexing and sonicating. Polyethylene glycol200, in the amount 4.495 gm, and 1N NaOH (0.173 gm) were then added.This mixture, in which the sodium dantrolene was in true solution (e.g.dissolved), was then loaded into 1 ml sterile syringes for injection,and used successfully in live animal tests.

For a dantrolene formulation made according to this protocol, a 240 mgdose of dantrolene sodium could be delivered in a volume of only about 7ml. Dimethylacetamide is currently used in one injectable product, andanimal tests conducted to date suggest that this formulation is safe forinjection as defined herein.

Example 3

A colloidal dispersion of dantrolene sodium at 5 mg/ml was prepared byfirst overlaying 0.101 gm of dantrolene sodium with 20 ml of an aqueoussolution of benzalkonium chloride, made by mixing 0.319 gm ofbenzalkonium chloride in 100 ml of distilled water; the dantrolenesodium is therefore at a level that greatly exceeds the solubility inwater (less than 0.4 mg/ml), and nearly all is dispersed as opposed todissolved. The mixture was then homogenized with a Polytron homogenizerat high speed for 3 minutes, to yield submicron particles. Zetapotential measurements using a Beckmann-Coulter Doppler ElectrophoreticLaser Scattering Analyzer (DELSA) showed a zeta potential of +28 mV.Clearly this charge is due to the adsorption of a stabilizing layer ofbenzalkonium chloride, since the dantrolene sodium would, of course,yield an anionic charge. Benzalkonium chloride is FDA approved insafe-for-injection products.

Example 4

In this Example, a phospholipid-based lyotropic liquid crystal was usedas a stabilizing layer on dispersed crystals of dantrolene sodium, againat the high concentration of 50 mg/ml. This was prepared by firstpreparing a “cubic phase” lyotropic liquid crystal, by mixing 1.595 gmof high-PC soybean phospholipid (Epikuron 200, from Lucas-Meyer, Inc.),1.121 gm of alpha-tocopherol, and 0.788 gm of distilled water. An amount0.349 gm of dantrolene sodium was mashed into the liquid crystal. To0.999 gm of this mixture was added 20 ml of the solution of benzalkoniumchloride described in Example 3, and the mixture was then homogenizedwith a Polytron homogenizer at high speed for 3 minutes, yieldingsubmicron particles. Zeta potential measurements using aBeckmann-Coulter Doppler Electrophoretic Laser Scattering Analyzer(DELSA) in this case showed a zeta potential of +72 mV. There was noevidence of a peak at +28 mV, as in the previous Example, thusindicating that the dantrolene crystals were coated withphospholipid-rich material, which in turn had an outer surface rich inbenzalkonium chloride.

Example 5

We tested two novel low volume dantrolene formulations as rescue agentsin porcine malignant hyperthermia. Each formulation was a low volumecolloidal suspension that dissolves readily upon injection into theblood stream. Formulations of both dantrolene sodium and dantrolene freeacid were evaluated as potential less cumbersome alternative treatmentarticles to Dantrium® IV that can be made immediately available forsingle bolus dose injection in volumes less than 10 ml for an adult.

The primary goal of this study was to evaluate the efficacy of lowvolume colloidal suspension dantrolene in the treatment of the crisis ofmalignant hyperthermia in malignant hyperthermia susceptible swine. Wehypothesized that both the sodium and free acid micronized dantroleneformulations would reverse the crisis of MH following bolus intravenousinjection of a weight based calculated treatment dose of 2.5 mg/kg.

This study, as well as both preliminary studies, were approved andperformed in accordance to the BAS Evansville Institutional Animal Careand Use Committee. Each study was performed as a randomized, open labelcomparison study performed at a single study center by the sameinvestigators.

In the first preliminary study, two dantrolene sodium and free acidformulations were evaluated for both safety and efficacy in 12 SpragueDawley rats. Prior to dosing, body weights were obtained and each ratreceived a single bolus dose injection of their respective test articlevia the lateral tail vein. All animals were observed immediately postdosing and continuously up through 30 minutes and again at approximately1, 2, 3, 4, 7, and 24 hours, prior to necropsy.

Example 6

In the second preliminary study, ten non-MHS domestic swine (Yorkshirecrossbreed) were used to determine the relative efficacious dose ofsingle formulations of dantrolene sodium and free acid capable ofcreating muscle relaxation. The methods as originally described byNelson and Flewellen, were followed, absent a sophisticatedmuscle-tension force measuring device. All swine were housed inaccordance with AALAC principles, acclimated at least 5 days prior tostudy in individual runs, fed twice daily with water ad libitum, in anisolated, temperature and humidity controlled room with a filtered airsupply with 12 hour cycled light. Animals were fasted 6 hours prior todosing. Each pig was pre-medicated with atropine sulfate (0.5 mg/kg),ketamine HCl (20 mg/kg), xylazine (2.5 mg/kg) and aceproxazine maleate(0.2 mg/kg). Intravenous access was established cannulation of anappropriate ear vein. Each animal then received thiopental (10.0 mg/kg)and were subsequently endotracheally intubated.

Once stable, each pig received its respective dose of either dantrolenesodium or free acid in a dose escalating fashion. Initial dose of 1.0mg/kg iv was administered, followed every two minutes by repetitive 0.5mg/kg bolus doses with the exception of one pig that received additivebolus doses achieving a cumulative dose of 10.5 mg/kg. Muscleresponsiveness to the relaxant effects of dantrolene was monitored viatrain of four (TOF) of the forelimb using a TOF Guard. The stimulus wasdelivered at 20 millivolts at 0.5 second intervals. Train of four wasmonitored for each dose level until muscle contraction was no longerevident in response to stimuli. At the conclusion of the study, each pigwas euthanized while anesthetized with intravenously delivered sodiumpentobarbital solution.

Data from this study was analyzed. The relative ED 95 for each thesodium and free acid formulation was determined to be 2.5 mg/kg as aweight based dose and advanced for study in the MHS swine as set forthin Example 7.

Example 7

In this study of the effects of two low volume, high concentrationcolloidal dantrolene formulations in the treatment ofhalothane/succinylcholine induced Malignant Hyperthermia in swine, nineswine that were shown by DNA analysis to be homozygous for the halothanesensitive allele (i.e., the 11 genotype) were studied. On the initialday of the study period, pigs were randomly assigned to the followinggroups:

Test Dose Dose Conc. No. of Animals Group Article mg/kg mg/mL (maximum)Control 0.9% saline 0 0 3 DFA (dantrolene free acid) 2.5 40 3 DS(dantrolene sodium) 2.5 50 3

Each of the pigs was anesthetized with 1M injections of atropine sulfate(0.05 mg/kg), ketamine HCl (20 mg/kg), xylazine HCl (2.5 mg/kg) andacepromazine maleate (0.2 mg/kg). Sodium thiopental (10 mg/kg) andintravenous fluids (0.9% saline; approximately 4.0 mL/kg/hr) wereadministered via a catheter into an ear vein. Animals wereendotracheally intubated and artificial ventilation was initiated.Endotracheally intubated animals were ventilated to ensure adequateoxygenation. The anesthetized animals were monitored for end tidalcarbon dioxide (ETCO2), intra-arterial blood pressures, peripheraloxygen saturation (SpO2), electrocardiograms, and core body temperature.

Following stabilization, administration of halothane 2% (approximately2MAC) was initiated. After approximately 15 minutes of halothaneadministration, succinylcholine (2 mg/kg) was administered via acatheter into an ear vein. Definitive diagnosis of MH crisis wasdetermined by the documented presence of at least two of the followingparameters: ETCO2>70 ton, increased rectal temperature >3° C., arterialpH of equal to/less than 7.25 and/or significant muscular rigidity.Following documentation of the onset of MH halothane was discontinued.Pigs either received no treatment (control) or one of the test articles(DFA or DS) via intravenous administration at a dose equivalent to theED95 (2.5 mg/kg) established in a previous study in normal pigs.Progression and/or regression of the MH crisis was evaluated atapproximately 1 minute intervals for the initial 20 minutes followingonset and then at 2 minute intervals until cessation (if attained).Neuromuscular blockade was monitored by measuring train of four (TOF)twitch in one of the forelimbs using a TOF Guard. The stimulus for theTOF was delivered as a train of four pulses where each pulse was 0.5seconds apart. However, reliable measurement of TOF was not possible asthe twitch response was masked by the profound muscle rigidity.Following treatment, the surviving pigs (DFA and DS) were allowed torecover from anesthesia and were euthanized at approximately 120 hourspost treatment.

All of the pigs developed MH after exposure to the triggering agents,halothane and succinylcholine. Typical signs of the MH episodes includedincreased core temperature, hypercarbia with ETCO2>70 mm Hg, an acidoticstate reflected by consistent decreases in arterial pH, significantmuscular rigidity, severe tachycardia and marked hypotension. Theconstellation of muscular rigidity, tachycardia and hypotension resultin state of hypoperfusion as evidenced by narrowing of the pulsepressure. After it was determined an MH crisis was observed, the pigsreceived either no treatment (control) or one of the test articles (DFAor DS). The control pigs were euthanized after it was determined thatthe MH episode was not naturally regressing. After treatment with DFA orDS, the MH crisis was quickly aborted in all animals. The pigs wereremoved from the ventilator, extubated, returned to their cages, andallowed an approximate 120 hour recovery period. Upon observation 12 to24 hours after return to their cages, there were no signs of cognitive,neurologic, or neuromuscular dysfunction in any of the treated animals.All of the treated pigs were judged by the principal investigator to benot remarkable at the terminal sacrifice.

Example 8

The pigs in Example 7, upon observation 12 to 24 hours after return totheir cages, had no signs of cognitive, neurologic, or neuromuscularfunction in any of the treated animals.

The phenomena of altered cognitive abilities and function as well asneuropsychiatric changes with or without impaired motor function iscommonly referred to as “pumphead” among anesthesiologists,cardiothoracic surgeons, and certain other medical personnel. Pumpheadis not related to MH. However, the inventors note that patients with MHhave an altered blood flow where the flow rate is not zero, but issignificantly different from normal. For altered blood flow thatrepresents a reduction in pressure, this is considered to be greaterthan a 10% decrease from baseline systolic pressure, or associateddecrease in mean arterial pressure, but less than a 95% decrease.Pulsatile changes or temporary elevations in blood pressure are alsoconsidered to be altered blood flow. In view of the observed results,the inventors envision that the prophylactic administration ofdantrolene, or one of its salts, analogs or relatives, preferably in lowvolume, high concentration form as described in Example 7 or,alternatively, in the normal form commonly used in the clinic anddescribed in the Professional Product Labeling for Dantrium® Intravenous(P&G Pharmaceuticals), should prevent or limit the effects of pumphead.While not being bound by theory, dantrolene may prophylactically addressneurological complications of pumphead via a unique and synergisticcombination of a number of intracellular and/or metabolic mechanisms,which work in concert for the stabilization of intracellular calcium andother concomitant actions. Dantrolene should also be suitable as atreatment capable of minimizing neurological complications when providedin a manner timely to an insult.

Cardiothoracic surgeons have, for many years, been performing open heartsurgeries for blocked coronary arteries, valve reconstruction, repair ofaortic arches and aneurysms, as well as other operations requiringcardiopulmonary bypass. While successful surgical outcomes are commonplace, so too are the deficits of memory, concentration, attention, andaffect that accompany procedures requiring cardiopulmonary bypass. Theincidence of the neurocognitive deficits is quite high. Publishedreports reveal that just over 50% of all CPB patients experience someform of cognitive deficit following surgery. A total of almost 35% ofpost-bypass patients continue to exhibit deficits at 6 weeks, and 24%suffer from deficits at one year post-bypass. The reported incidence ofneurocognitive deficit attributed to CPB is approximately 54% at 5 yearspost-bypass. The exact nature and etiology of neurocognitive deficitsassociated with CPB is not completely understood, but has been wellstudied in a number of controlled prospective studies.

Neurocognitive deficit induced from iatrogenic insult, such as in thecase of “pumphead” arising from cardiopulmonary bypass, or traumaticincidents reflects a complex and multifaceted injury. Some researchershave suggested that neuronal injury can occur in response to vagueconditions such as hypoxia, ischemia, insufficient glucose levels, orinappropriate blood pressures or insufficient flow rates or pulsatilepressures. Individually, a description can be proposed for the cause andeffect for various factors and their potential relationship to neuronalinjury or neuronal death. For example, it is known as fact that a givenmass of “cool” cerebral tissue has a lower energy demand and, hence,consumes less oxygen and glucose than the same given mass at bodytemperature. It is also true that one principle reason for coolingcerebral tissue while on cardiopulmonary bypass is to decrease themetabolic demands of this tissue. The concept is that cool tissue willbetter survive the sub-optimal supply of blood, oxygen, glucose andother nutrients as well as the decreased ability to metabolize andeliminate physiologic waste products while on CPB. Further, it is likelyto be true that upon re-warming from a cooled “neuroprotective” state,the physiologic requirements of individual cells may well exceed thesupply of oxygen and nutrients than can be delivered under the normalflows of CPB. These, however, do not elucidate the underlying mechanismbehind the resulting neurocognitive deficit (pumphead), nor reveal thebest method of treatment.

Other researchers have implicated specific ion channels or receptors;such as NMDA, non-NMDA ionotropic, sodium channels, calcium channels andothers as potential causes for neuronal injury or death. Still othershave cited the presence or deposition of substances such as glycine,glutamine, glutamic acid, kainic acid, and others as possible toxicagents.

An array of potential receptor-mediated biochemical mechanisms have beendiscussed in the literature as possible explanations for the centralorigins of pumphead. Essentially, each of the various schools of thoughthas had its favored mechanism, usually centered around a particularreceptor—or even a particular subunit of a particular receptor, as inthe case of Chenard, U.S. Patent Application Pub. No. 2002/0072485 andothers by Chenard, or Kozachuk, U.S. Patent Application Pub. No.2003/0045450.

Thus, one school of thought has focused on N-methyl-D-aspartate (NMDA)receptors, which can mediate flow of calcium ions into the cell from theextracellular space. This school of thought, typified by the Chenardapplication, holds that an effective means of protecting againstneurological damage from impairment of glucose and/or oxygen supply tothe brain is simply to treat with an NMDA antagonist, of proper subunitselectivity. However, it is a fairly easy matter to find reasons whysuch a simplistic approach might be doomed to failure, reasons thatinclude the theory of calcium-induced calcium release (CICR), whichholds that even a relatively minor increase in intracellular calciumions can trigger the release of calcium from the endoplasmic reticulum.See for example Makarewiez et al., J. Neurochem., 85(suppl. 2):20. Thecurrent inventors recognize this as a consequence of an intact ryanodinereceptor mechanism, which provides an ample intracellular source—theER—for calcium ions, either in the face of incomplete blockage of theNMDA-R mechanism or of other pathways to calcium ion influx. In short,in view of the CICR mechanism, nearly complete blockage of the NMDA-Rmechanism would be required to prevent triggering of intracellularcalcium ion release, and even if complete blockage were accomplished,other pathways for calcium-induced calcium release from intracellularstores would need to be blocked in any case.

Another school of thought focuses on glutamate receptors that arenon-NMDA receptors but which can also mediate flow of calcium ions intothe cell. See for example Bokesch, Am. Soc. Anesth. Newsletter, 1996,vol. 60 (8). In the context of the aforementioned, CICR, this quitelikely represents another parallel mechanism for triggering of calciumrelease, which cannot be blocked through NMDA-R antagonism.

Yet another school of thought focuses on the kainic acid (KA) mediatedmechanisms, in the context of apoptosis of neuronal cells. Thus,kainic-induced neurological damage was prevented byalpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)receptor antagonist CNQX, but not by the NMDA receptor antagonist MK801,nor by the membrane L-type calcium channel antagonist nifedipine. See LiS Y, Ni J H, Xu D S, and Jia H T, Neurosci Lett. 2003 Dec. 4;352(2):105-8. In other words, in contrast with the NMDA school ofthought, these authors found evidence of a calcium ion-related mechanismof neuronal damage that is not treatable by simply applying an NMDAantagonist.

Currently, drugs are under development that target every step in thecascade of events contributing to neuronal damage and cognitive loss.These include glutamate-release inhibitors, NMDA receptor antagonists,sodium and calcium channel blockers, free radical scavengers, apoptosisinhibitors and others. What does not seem to have been recognized andfocused upon is the multiplicity of mechanisms by which neuronal damageand cognitive loss can occur with altered blood flow and changes in bodyor tissue temperature—even under the umbrella of Ca2+ mediatedmechanisms—and thus the conclusion that a number of these mechanismsmust be blocked in concert has not been recognized, nor has therequisite pharmacological intervention been elucidated in light of thisconclusion. In particular, what also does not seem to have beenrecognized is the significant therapeutic potential of dantrolene andits simultaneous pharmacologic activities against a number of thesemechanisms. The current invention emphasizes that pharmaceutical-basedprophylaxis and treatment of “pumphead” and related injuries should havedantrolene as the primary modulator of intracellular calcium; althoughcombinations of dantrolene with other agents are within the scope of theinvention, using anything other than dantrolene (or a salt, analogue orrelative thereof, which is a ryanodine receptor antagonist) will,broadly, lower the therapeutic index and/or result in sub-optimalprevention or treatment.

That dantrolene blocks the release of intracellular calcium stores fromthe endoplasmic reticulum is well understood. However, in separatepublications from distinct groups, dantrolene has been shown to be aneffective inhibitor, either directly or indirectly, of at least threeadditional mechanisms affecting neuronal damage and cognitive function.Evidence from cell culture studies by Frandsen and A Schousboe (Journalof Neurochemistry, Vol 60, 1202-1211) shows that dantrolene inhibits thetoxicity induced by both glutamate and NMDA. Also in cell culture,Frandsen and Schousboe also showed that the toxicity of quisqualate(QA), which stimulates Ca2+ release from an intracellular store that isindependent of Ca2+ influx, is also inhibited by dantrolene. Moreover, a2002 publication from Romanian researchers (Popescu et al., J. Cell.Mol. Med. 6(4):555) showed that dantrolene inhibits the kainicacid-mediated apoptosis mechanism.

The current inventors recognized for the first time that dantroleneadministration provides at least four synergistic protective actions inthe context of altered blood flow scenarios which are simultaneouslyrequired for neuroprotection in the case of cardiopulmonary bypass andagainst other iatrogenic cerebrospinal disturbances. Thus, the currentinventors have recognized that neurocognitive and motor deficits whichare experienced by some patients after anesthetics and operationsutilizing extracorporeal circulation, such as CPB, or in case whereinduced hypotension or hypothermia is performed, are the result of aconstellation of factors, with no one event or factor being singularlydominant as the causative factor, and yet dantrolene has the uniqueability to treat multiple mechanisms in such a way as to provide broadprotection in these circumstances.

Conclusions drawn from studies of the peripheral nervous system, or fromcranial nerves such as the optic nerve, are broadly of questionablevalue in matters of pumphead, and of cerebrospinal tissues in general.Underscoring this is the fact that surgical patients with a medicalhistory that includes stroke are no more likely to suffer from pumpheadthan those with no stroke history. See Warner, Int. Anesth. Res. Soc.2004 Review Course Lectures, presented at the 78th Clinical andScientific Congress, Tampa, Fla., p. 123. One basis for understandingthis lies in the physiology of the cranial nerves as contrasted with thecerebrospinal nervous tissue.

In particular, the twelve paired cranial nerves, with the exception ofCN I (olfactory) and II (optic), originate in the brainstem; which iscomprised of the midbrain, pons, and medulla oblongata. Cranial nervesare generally categorized as being sensory, motor or mixed (both sensoryand motor). Cranial nerves originate at nuclei located on the brainstem,with sensory nuclei located laterally and motor and mixed nuclei morecentrally located. The sensory nuclei receive their sensory input fromthe periphery, but the sensory receptor cell bodies are never in thenucleus itself. Rather, they are located just outside the CNS inganglion.

Cranial nerves, as PNS components, tend to be accompanied by a dedicatedarterial blood supply that, via smaller perforating arteries, provideblood flow throughout its length. Typically, cranial nerves lack anysignificant source of collateral blood flow. As an Example, the opticnerve has an average diameter of 1.5 mm and has an intra-orbital lengthof about 30 mm and maintains a dedicated vessel throughout its entirelength. The ophthalmic artery arises from the distal end of the internalcarotid artery and travels with the optic nerve toward the posterioraspect of the eye. The posterior third of the optic nerve is supplied byvessels arising from the anterior communicating and anterior cerebellararteries, while the anterior two thirds of the nerve is supplied by thecentral retinal artery. Occlusion of this arterial conduit will resultin a decrease or total cessation of blood flow to the tissues of thisorgan, including the neural cells. A specific Example of the effects ofsuch an ischemic event is evidenced in the condition known as amaurosisfugax. Here, the central retinal artery is partially or totally occludedby an embolus (or emboli) resulting in transient (or longer lasting)monocular blindness or other disturbances of visual field recognition.

In contrast to cranial nerves, the many sensory and motor tracts of thespinal cord tend to receive their blood supply via multiple vessels withabundant collateral circulation. Throughout the cervical and thoracicregions, the spinal cord receives the bulk of its blood flow via asingle anterior spinal artery and two posterior spinal arteries as wellas collateral supply from branches from the intercostal arteries and thedescending thoracic and lumbar aorta. The nature of the blood supply tothe spinal cord minimizes the likelihood of ischemia from episodicembolic phenomena.

In instances of trauma with cord and arterial compression, or in casessuch as surgical aortic cross clamp during aneurismal repair,insufficient blood flow to the cerebro-spinal cord can occur and lead tocertain neurologic insults. This is especially evident in operationsduring which blood flow to the lower third of the cord via the artery ofAdamkiewicz (arteria radiculris magna) is compromised. The incidence oftransient post-operative deficits and post-operative paraplegia arereported to be 11% and 6% respectively. Higher rates are reported ascross-clamp time exceeds 30 minutes. The classic deficit is that of ananterior spinal artery syndrome with loss of motor function and“pinprick” sensation, with preservation of proprioception and vibrationsensation.

The role and relationship of non-normothermic states of body temperatureto the above is important. Altered states of temperature are easilyinduced by medical practitioners. Non-normothermic states of hypothermiacan be readily induced under general anesthesia both intentionally, asin cardiopulmonary bypass, or unintentionally, where appropriatesafeguards are not employed to guard against the loss of body heat.

A number of potential complications are associated with unintentionalintraoperative hypothermia including altered clotting function withincreased blood loss, increased frequency of infection and myocardialstress. As such, the routine practice of anesthesia has largely evolvedto practice the maintenance of normothermia during most operativeprocedures.

Little evidence exists today to show that intraoperative hypothermiaimproves outcome except in the instance of deep hypothermia forcirculatory arrest while undergoing cardiopulmonary bypass. Completecirculatory arrest for periods of up to one hour at core temperaturesranging from 16 degrees to 18 degrees C. offers some protection for theadult brain; where patients are expected to recover neurologically, butnot necessarily neurocognitively, intact. Otherwise, mild and moderatehypothermic conditions where temperatures typically range form 32degrees C. to 34 degrees C. have been evaluated in a number ofrandomized trials during CPB and have shown little, if any benefit tothe patient. The issue of employing mild to moderate hypothermia duringCPB as a neuroprotective technique is difficult to assess because itrequires not only reducing core temperatures but rapid re-warming cyclesthat usually delivers hyperthermic blood to the cerebrospinal system,which may negate any potential benefit that hypothermia may haveprovided

Mild to moderate hypothermia has been evaluated in a large prospectiverandomized trial as a potential therapeutic maneuver to treat patientswith traumatic brain injury while in the Intensive Care Unit. In thisstudy, no benefit was attributed to hypothermia and, in fact, elderlypatients suffered a greater rate of complications when randomly assignedto the hypothermic group.

The non-normothermic state of hyperthermia is a common sequellae ofacute brain injury. Animal studies have shown that temperatures rangingfrom as little as 1 degree C. from normal, while either during or aftervarious forms of acute brain injury markedly worsen neurologic outcome.The presence of hyperthermia has been regarded as a reliable prognosticindicator of poor neurological and neurocognitive outcome in acute braininjury. We know of no proposed advantages, theoretical or otherwise,linking hyperthermia to improved neurological or neurocognitiveoutcomes.

Regarding NMDA and non-NMDA receptors, it is likely that the act ofeither cooling, re-warming, or the cyclic combination of both coolingand re-warming of the cerebrospinal system results in the expression ofthese potentially destructive receptor mechanisms. It is also likelythat the temperature flux causes an imbalance of nutrient substratessuch as oxygen and glucose out of balance to the specific needs of thecerebrospinal system as any given moment in the course of the coolingand re-warming procedure.

The application of a single, safe agent, namely dantrolene or one of itssalts or relatives, for the prevention and treatment of neurological andcognitive damage in CPB and related insults has fundamental advantagesover combination approaches that could be envisioned. To begin with, thesafety record and therapeutic index of dantrolene sodium are extremelyfavorable. In the context of this patent, we define “therapeutic index”of a therapeutic drug (or mixture) to be the quotient A/B, where A and Bare defined as follows: A is the LD50 (dose yielding 50% lethality) ofthe drug when given intraperitoneally to rats; and B is the dose of thedrug that when given i.p. yields 50% reduction of apoptotic nuclei inthe cortex of rats given 5 mg/kg kainic acid, according to the protocoldescribed by Popescu et al. in [J. Cell. Mol. Med. 6(4):555 (2002)]. Asthe quantity A has been shown to be 780 mg/kg (Fournier, P, 1982,Dossier toxicologique, pharmacologique, pharmacocinetique du DantriumIV. Lyon, Laboratorie Obercal), and the value of B is 10 mg/kg accordingto the Popescu paper cited above, the therapeutic index for dantroleneas defined herein is calculated to be 78. A therapeutic index greaterthan 10, and especially greater than about 50, is viewed in the contextof this invention as being of importance, particularly in the context ofa surgical procedure where drug interactions are already complicated,and a large zone of comfort (at least an order of magnitude) betweenadministered dose and lethal dose is of course highly desirable. Forexample, dantrolene does not cause cardiopulmonary depression even atdoses as high as 7.5 mg/kg i.v. Such depression, if caused by either ofthe drugs in a given combination, would of course be potentiallydetrimental in the context of a cardiopulmonary bypass operation. Thisis certainly the case for suggested combinations involving localanesthetics (as sodium channel modulators), since the cardiotoxicity ofthe ‘caines (lidocaine, bupivacaine, etc.), and the low therapeuticindex, is well known. Only rarely does dantrolene cause severecardiopulmonary complications when combined with calcium channelblockers. In contrast, the drugs to be used in the combinationsdescribed in Jensen (U.S. Patent Application Pub. No. 2003/0092730), forExample, include drugs such as topiramate (Topamax), which “ . . . has apotential . . . to cause CNS depression, as well as other cognitiveand/or neuropsychiatric adverse events . . . ”. (2001 Physicians’ DeskReference, page 2394).

The prevention of cognitive loss—pumphead—due to CPB or relatedcircumstance differs in many fundamental ways from the treatment of apre-existing disease. It does not involve any known dominant heredity orother prefactor that introduces heightened risk of damage, as is thecase with Malignant Hyperthermia. Since the preventive steps are to betaken in the absence of a pre-existing neurological disorder, such stepsmust necessarily be highly safe, in order to comply with a reasonablebenefit/risk ratio. The increase in focus and certainty that comes fromthe diagnosis of a pre-existing condition is not present. And in thecurrent climate of medical practice, prevention typically plays asecondary role to treatment.

The neuroprotective efficacy of a low volume/high concentrationdantrolene formulation may be demonstrated using a recovery model of CPBin the rat described by Mackensen et al (Anesthesiology. 2001 December;95(6):1485-91). For example, three groups of rats may be subjected to 60min of normothermic (37.5 degrees C.) nonpulsatile cardiopulmonarybypass (CPB) using a roller pump and a membrane oxygenator. Group 1 rats(n=10) receive no treatment. Group 2 rats (n=10) are pretreated with lowvolume dantrolene IV, 2.5 mg/kg, and Group 3 rats (n=10) are pretreatedwith low volume dantrolene IV, 5.0 mg/kg. A fourth group, (Group 4)serve as sham operated controls (n=10). Neurologic outcome is assessedon days 1, 3, and 12 after CPB using standardized functional testing.Neurocognitive outcome, defined as the time (or latency) to finding asubmerged platform in a Morris water maze (an indicator ofvisual-spatial learning and memory), is evaluated daily from post-CPBdays 3-12. Under this investigation, the neurologic outcome should beworse in Group 1 versus the Groups 2, 3 and 4 at all three measurementintervals. Group 1 should also have longer water maze latencies comparedwith Groups 2, 3 and 4, indicating significant neurocognitivedysfunction after CPB. This investigation should demonstrate thatdantrolene pretreatment, at both 2.5 mg/kg and 5.0 mg/kg attenuates CPBassociated neurologic and neurocognitive impairment in a rodent recoverymodel.

The neuroprotective effect of dantrolene may be compared with that ofxenon, an agent previously shown to be protective in this animal model.(Ma et al, Anesthesiology. 2003 March; 98(3):690-8) In this comparison,following surgical preparation, rats would be randomly divided into fourgroups of 10 rats per group: (Group 1) sham rats would be cannulated butwould not undergo nonpulsatile cardiopulmonary bypass (CPB); (Group 2)CPB rats would be subjected to 60 min of CPB using a membrane oxygenatorreceiving a gas mixture of 30% O2, 65% N2, and 5% CO2; (Group 3)CPB+dantrolene rats receive dantrolene (10.0 mg/kg IV) 15 min prior toundergoing 60 min of CPB with the same gas mixture as Group 2; and(Group 4) CPB+xenon rats undergo 60 min of CPB using an oxygenatorreceiving 30% O2, 60% xenon, 5% N2, and 5% CO2. Following CPB, the ratswould recover for 12 days, during which they would undergo standardizedneurologic and neurocognitive testing (Morris water maze). In thisinvestigation, the sham, CPB+dantrolene and CPB+xenon groups all wouldhave significantly better neurologic outcome compared to the CPB groupon postoperative days 1 and 3. Compared to the CPB group, the sham,CPB+dantrolene, and CPB+xenon groups would have better neurocognitiveoutcome on postoperative days 3 and 4. By the 12th day, theneurocognitive outcome would remain significantly better in theCPB+dantrolene and CPB+xenon groups compared to the CPB group. Thisinvestigation would show the efficacy of dantrolene (10.0 mg/kg) inattenuation of CPB-induced neurologic and neurocognitive dysfunction iscomparable to xenon.

In humans the neuroprotective effect, e.g., effectiveness in preventingor reducing pumphead, could be demonstrated by an investigation wheretwenty patients about to undergo coronary artery re-vascularizationduring cardiopulmonary bypass would be randomly assigned to either adantrolene treatment or non-treated control group. Prior to surgery,each patient would be given a battery of nine standard tests designed tomeasure cognitive function in four broad categories; attention andconcentration; verbal memory; abstraction and visual orientation; andfigure (numbers) memory. Patients would again administered the sametests 24 hours and six weeks post-operatively. Each assessment would beperformed by the same investigator who would be blinded to the patient'sstudy group assignment. At the time of the operation, each patient wouldbe induced of general anesthesia according to a protocol utilizing amodified cardiac/narcotic technique. All agents would be administered ona weight based dose (mg/kg) whenever possible. Volatile anestheticagents would be administered and regulated by the anesthesiologist viathe endotracheal tube to maintain adequate blood and pulse pressuresboth pre and post bypass, and by the perfusionist during bypass tomaintain pressures suitable for adequate tissue perfusion. Astandardized protocol by which the operation is to be performed would bedesigned and applied to each patient enrolled in this study. Protocolsare developed for each aspect and phase of the operation, includingvena-caval/atrial cannulation; initiation and maintenance ofcardiopulmonary bypass utilizing a membrane oxygenator; initiation andmaintenance of cardioplegia; standardized monitoring, induction, andmaintenance of cooling and re-warming procedures; and recommendedprocedures for preparation for separation and actual separation fromcardiopulmonary bypass, including acceptable doses of inotropic/pressoragents and transfusion therapies. Patients randomized to Group 1(dantrolene) would receive 1.0 mg/kg of 5% (50 mg/ml) colloidaldantrolene via central venous access after the patient has beensuccessfully endotracheally intubated and stabilized of generalanesthesia, and prior to sternotomy. (For purposes of this particulartrial, a dose of 1.0 mg/kg is administered to each patient althoughdoses ranging from approximately 0.1 to 10 mg/kg and above are likely toprovide a neurocognitive protective effect). The entire dantrolene dosewould be administered over approximately 30 seconds. In order to ensurethe double blind nature of this study, either the low volume, highconcentration colloidal dantrolene (5%) or a placebo control solution ofcomparable volume would be injected at the appropriate time by the studycoordinator. The anesthesia and surgical staff would remain blinded tothe treatment assignments. Upon completion of the operative procedure,patients would be treated via standard post-CPB “fast track” treatmentprotocols whereby they are endotracheally extubated in the operatingroom upon emergence or within six hours of arriving in the Cardiac PostAnesthesia Care Unit. Approximately 24 hours and 6 weeks post extubationpatients would be administered the same battery of the nine standardizedtests in the same order and fashion as performed pre-operatively. Toreduce possible inconsistencies of interpretation, assessments at eachtime interval would be performed by the same blinded investigator. Insuch an investigation, colloidal dantrolene treated patients wouldexhibit significantly less neurocognitive dysfunction than untreatedpatients. The findings would be significant for the 24 hour post-opassessment and for the six week follow-up assessment. Furthermore,patients receiving dantrolene therapy would test significantly betterthan control patients in those tests designed to assess attention andconcentration. Again, the results would be similar for both post-opevaluation periods. The study would demonstrate that dantrolene, 1.0mg/kg attenuates CPB-induced neurologic and neurocognitive impairment inman.

In this patent, we put forth the use of dantrolene and its salts,analogs and relatives for the prevention of neurological andcerebrospinal injury in a number of conditions that have not previouslybeen recognized as treatable by this medication, nor any othermedication for that matter. The invention applies in relation to anumber of specific factors that induce a state of low systemic bloodflow or decreased cerebral perfusion pressures, and puts forth the useof dantrolene as a preventive means. These would include, but notnecessarily be limited to the following examples:

1) extracorporeal oxygenation and perfusion systems commonly utilized incardiopulmonary bypass for thoracic and coronary artery bypass graftingsurgeries (CPB), as well as other enabling techniques such as deephypothermic circulatory arrest allowing for complex reconstructive openheart procedures such as aortic arch repair/replacement in neonatal,pediatric and adult patients where minimal blood flow (approximately 90%of normal) is generated. Neurologic complications are reportedly as highas 54% in those having undergone CPB for coronary artery bypass grafting(CABG) and other related thoracic operations [Warner, op. cit.].Neuropsychiatric alterations range from subtle to severe cognitiveimpairment, personality changes, delirium, memory loss, and organicbrain syndromes. Some patients experience transient and/or permanentimpaired motor function. Estimates of patients sustaining permanentdeficits range from 2% to 50% or more. The risk of neuropsychiatricinjury tends to increase as the total length of CPB time increases.Shorter periods of CPB are, however, not necessarily risk free and arealso known to cause neuropsychiatric and cognitive alterations. In theinstance of CABG performed without the use of extra-corporealoxygenation and perfusion (off-pump techniques), patients reportedlyhave experienced signs and symptoms associated with “pumphead”. This isbelieved in part to be due to periods of controlled (induced)hypotension (decreased cardiac stoke volume and cardiac output) and/orinduced bradycardia (decreased heart rate) established to createconditions suitable for coronary graft placement and suturing. Thenormal cardiac cycle results in systemic blood flow which is pulsatilein arterial vessels. The arterial tree continues to taper in diameterand, upon reaching the systemic capillary blood vessels in tissues andend organ beds, the pulsatile flow gradually changes to a continuousflow, also known as laminar flow. CPB establishes a decreased systemicblood pressure, decreased mean arterial pressure (MAP) as well as adecreased pulsatile waveform pattern of blood flow normally generated bythe usual cardiac cycle of contraction and relaxation, which yieldsspecific and independent systolic and diastolic pressures. Whilecontroversy continues to surround the “ideal” systemic arterialpressures to be generated by CPB and other extracorporealoxygenation/perfusion systems (as read by radial artery arterialpressure tracing), the majority of heart centers and perfusioniststypically recommend and practice generating CPB flow rates of 2.0 to 2.5L/min/m2 (approximately 50 to 60 ml/kg/min) which will usually generatea mean arterial pressure between 50 and 80 mm Hg.

2) surgical procedures using extracorporeal intervention in blood flowother than CPB, including but not limited to extra-corporeal membraneoxygenation (ECMO), states associated with the induction and maintenanceof induced and/or controlled hypotension as commonly employed inneurosurgery, vascular surgery and “off-pump” coronary artery bypassgrafting surgery. Dantrolene treatment and/or pretreatment is recognizedin this patent as preventive in the case of other conditions, sinceneuropsychiatric changes, altered cognitive function, and impaired motorfunction are not solely related to decreased pressures and flow ratescaused by CPB. Extracorporeal membrane oxygenation (ECMO) is arelatively new treatment modality which provides for a temporizingmethod of extracorporeal oxygenation in patients, typically neonates,whose lungs cannot withstand more conventional mechanical or assistedventilation techniques. This particular patient population experiencesan unusually high risk of cerebral, cognitive, and motor impairment.

certain trauma conditions, especially shock and trauma associated withdecreased intravascular circulating blood volumes, and particularlyinjuries associated with increased intracranial pressures (ICP),decreased cerebral blood flow (CBF) and altered cerebral perfusionpressures (CPP). Importantly, conditions treatable by dantrolene as perthis patent include trauma to the central nervous system, especiallyevents resulting in head injuries. In either closed or open head trauma,the brain typically sustains injury on a number of levels and in acascading fashion. These injuries are frequently accompanied withincreased intracranial pressures attributed to cerebral hemorrhagicevents or to advancing cerebral edema. As intracranial pressures (ICP)increase (due to edema or hemorrhage), the autoregulated cerebral bloodflow is further impaired both locally and globally. Arterialhypertension occurs as a result of inborn physiologic reflexes, whichfurther aggravates cerebral edema and increases ICP. Cerebral perfusionpressure is defined as the difference between mean arterial pressure atthe level of the brain and either the central venous pressure or theintracranial pressure, which ever is greater. It is widely recognizedthat this pressure should be maintained above 60 mm Hg in order tosustain adequate CPP, cerebral perfusion and cerebral blood flow.Maintaining adequate perfusion pressures may be difficult if notimpossible, in the setting of many head injuries. Brain injury,especially injuries associated with compromised cerebral blood flow fromaltered CPP and increased ICP, is frequently associated withneurophysiologic alterations as well as impaired cognitive and motorfunction. It is further anticipated that due to local conditionsestablished by reflexes similar to those described above as related tohead injuries, that the long term effects of spinal cord injury may beminimized or in someway ameliorated by the administration of dantrolene,one of its salts, analogs or relatives.

The invention also applies in relation to non-normothermic temperaturesresulting from induced hypothermia techniques utilized as a possibleneuroprotective measure or as a function of deep circulatory arrestwhile on CPB as well as the re-warming periods and possible hyperthermicovercorrection, and hypothermia resulting from the poikilothermic natureof anesthetized patients, as well as episodic hyperthermia resultingfrom exogenous or endogenous influences, including but not limited tosepsis, hypothyroidism, hemorrhagic brain injury, overaggressiveattempts to rewarm, and fulminant infection.

For “pumphead” and the related applications of focus in this Example,the currently marketed dantrolene formulations may be applicableprovided that the large volumes of administration are not prohibitive,as may be the case in many clinical situations (though less commonlywith field situations), and where the mannitol present in such aformulation is not strongly contraindicated. Both oral and injectableDantrium® formulations (Procter & Gamble) can be used prophylactically,and in particular the injectable Dantrium® formulation is applicableeither prophylactically or therapeutically.

It can be readily envisioned that a dantrolene salt, in apharmaceutically acceptable formulation, can be administered asprophylactic treatment by skilled practitioners, prior to inducing analtered physical or physiologic state via some form of medical orsurgical intervention known to compromise, or in some way potentiallyjeopardize, the baseline neuropsychiatric state and cognitive functionof any one individual. Furthermore, it is also expected that treatmentwith such a formulation would yield benefit in the treatment ofalterations in neuropsychiatric or altered cognitive abilities whentreatment is initiated in a timely fashion, when deficits may beattributed to any number of factors as mentioned above.

It is anticipated that a wide range of doses of this dantrolene sodiumformulation will obtain the intended effect, particularly in view of thehigh therapeutic index of dantrolene. A lower volume formulation asprovided herein will allow for easier and more accurate administrationin a more rapid manner.

It is expected that doses ranging from 0.1 to 10.0 mg/kg in single ordivided multiple doses will prove efficacious, depending upon the age,pre-existing state of health, and possible extent of neurologic injury,and depending upon the type and extent of the insult. The preferredrange is about 0.5 to about 4 mg/kg, as a single, total dose. Multipledoses or extended dosing schedules may be employed depending upon thenature or duration of the underlying physiologic insult.

In addition to dantrolene salts, other agents may provide similarprotection against neuropsychiatric changes and cognitive impairment,particularly in cases where the agent has similar pharmacologic actionas dantrolene sodium, and especially if it is known to provide relieffrom MH. Thus, a pharmacologically active relative of dantrolene, suchas a compound containing a hydantoin group and/or a nitrophenyl ornitrofuranyl group, which affects the ryanidine receptor and through itintracellular calcium release, would be expected to be active within thepresent invention, particularly if it diminishes the symptoms of MH. Asan instructive example, while certain analogues such as azumolene arepharmacologically related to dantrolene and may be of use in the presentinvention, dantrolene would be preferred over azumolene because thelatter has been shown to be of limited benefit in the treatment ofMalignant Hyperthermia (MH); in contrast, dantrolene sodium is the mostefficacious rescue agent known for MH. It is also anticipated that newdantrolene analogs and chemical relatives will become available, and tothe extent that such a new agent has similar pharmacologic actions, andespecially to the extent that it relieves the symptoms of MH, it is tobe expected that the same agent can be used in the context of thepresent invention.

The present invention also provides dantrolene sodium in apharmaceutically acceptable formulation that can deliver the requisiteamount of drug in a liquid volume that is one or, in some embodiments,two orders of magnitude less than that required by the current Dantrium®formulation (which requires volumes on the order of one-half to oneliter for a human application), and which therefore minimizes orcircumvents the complications and dangers associated with large liquidvolumes of administration, particularly for the treatment of theconditions of focus in this patent, including but not limited tomalignant hyperthermia and pumphead. This substantial reduction involume and associated problems is not foreseen in the Mangat et al.patent, but should be considered of high importance in view of, forexample, the added complications imposed on the surgical team when aliter of aqueous solution must be administered in a procedure whosesuccess is dependent on critical control of an extracorporeal circuit.Furthermore, the sheer time required to reconstitute several dozen vialsof the currently marketed I.V. dantrolene formulation can have severerepercussions in the attempted treatment of many of the CNS disturbancesof focus herein, particularly emergency situations. With certainembodiments of the current invention, a dantrolene dose of up to 500 mgcan be delivered in liquid volumes less than 50 ml in all cases; a 300mg dose can be delivered in a volume of less than 30 ml, more preferablyless than 10 ml, and most preferably less than or equal to about 5 ml.The latter volume is sufficiently small that the entire formulationcould be loaded into an auto injector in accordance with standardvolumes of such devices.

Certain embodiments of this invention, exemplified but not limited tothe Examples herein, provide low-volume dantrolene sodium formulationsthat are either a solution, or contain particles that are sufficientlysmall to permit safe intravenous injection, in particular such that over95% of the particles are less than 0.8 microns, or preferably less than0.45 microns (viz., such that they can pass through a standard 0.45micron filter). Other routes, such as intramuscular, intrathecal,intraocular, extracorporeal, etc. are also made possible by these lowvolumes of administration.

Low-volume formulations of dantrolene and its salts can be prepared in anumber of ways. The pharmaceutically acceptable solventN,N-dimethylacetamide, together with hydroxyl-containing solvent(s),provide for a powerful solubilization matrix, and this can be modulatedwith polyethylene glycol (PEG), and appropriate modifiers such as baseand surfactant. Alternatively, small particles of solid dantrolene orone of its salts can be dispersed by homogenization techniques, forexample, as described in Examples 1, 3 and 4.

What is claimed is:
 1. A method for treating heat stroke in mammal,comprising the step of: administering to a mammal in need of treatmentfor heat stroke a therapeutically effective amount of a safe forinjection, low volume liquid formulation of dantrolene sodiumcomprising: dantrolene sodium at a concentration in the range of 3.33mg/ml to 166.67 mg/ml; a water-soluble polysorbate; a compound selectedfrom the group consisting of sorbitol and mannitol; and water as aliquid carrier, wherein said dantrolene sodium and water are presenttogether as a colloidal dispersion of dantrolene sodium particles in thewater, wherein the dantrolene sodium particles are less than about 2microns in average diameter, and wherein the formulation is safe forintravenous administration.
 2. The method of claim 1, wherein theformulation comprises dantrolene sodium at a concentration in the rangeof 30-80 mg/ml or in the range of 10-60 mg/ml.
 3. The method of claim 1,wherein the formulation consists essentially of: dantrolene sodium at aconcentration in the range of 3.33 mg/ml to 166.67 mg/ml; awater-soluble polysorbate; a compound selected from the group consistingof sorbitol and mannitol: and water as a liquid carrier, wherein saiddantrolene sodium and water are present together as a colloidaldispersion of dantrolene sodium particles in the water, wherein thedantrolene sodium particles are less than about 2 microns in averagediameter, and wherein the formulation is safe for intravenousadministration.
 4. The method of claim 3, wherein the formulationcomprises dantrolene sodium at a concentration in the range of 30-80mg/ml or in the range of 10-60 mg/ml.
 5. The method of claim 1, whereinthe dantrolene sodium is the primary modulator of intracellular calciumpresent in the formulation.
 6. The method of claim 1, wherein theformulation further comprises polyvinylpyrrolidone (PVP).
 7. The methodof claim 6, wherein the formulation consists essentially of: dantrolenesodium at a concentration in the range of 3.33 mg/ml to 166.67 mg/ml; awater-soluble polysorbate; a compound selected from the group consistingof sorbitol and mannitol; polyvinylpyrrolidone (PVP); and water as aliquid carrier, wherein said dantrolene sodium and water are presenttogether as a colloidal dispersion of dantrolene sodium particles in thewater, wherein the dantrolene sodium particles are less than about 2microns in average diameter, and wherein the formulation is safe forintravenous administration.
 8. The method of claim 7, wherein theformulation comprises dantrolene sodium at a concentration in the rangeof 30-80 mg/ml or in the range of 10-60 mg/ml.
 9. The safe method ofclaim 1, wherein the compound is mannitol and the formulation comprisesno more than 30 milligrams of mannitol per milligram of dantrolene. 10.The method of claim 1, wherein the administering step further comprisesadministering a quantity of 3-150 milliliters of the formulation to themammal.
 11. The method of claim 10, wherein the quantity is 10milliliters or less.
 12. The method of claim 11, wherein the quantity is5 milliliters or less.
 13. The method of claim 1, wherein theadministering step further comprises administering a dose of 250-300 mgdantrolene sodium to the mammal.
 14. The method of claim 10, wherein theadministering step further comprises administering a dose of 250-300 mgdantrolene sodium to the mammal.
 15. The method of claim 1, furthercomprising a step of: preparing the safe for injection, low volumeliquid formulation of dantrolene sodium by combining a dry formulationcomprising: dantrolene sodium consisting essentially of dantrolenesodium particles less than about 2 microns in average diameter; awater-soluble polysorbate; and a compound selected from the groupconsisting of sorbitol and mannitol, said dry formulation beingreconstitutable by water to provide a colloidal dispersion of dantrolenesodium particles less than about 2 microns in average diameter in thewater that is safe for intravenous administration, with water to form aliquid formulation that is a colloidal dispersion of dantrolene sodiumparticles less than about 2 microns in average diameter in the waterthat is safe for intravenous administration, and in which the dantrolenesodium is present at a concentration in the range of 3.33 mg/ml to166.67 mg/ml, and whereupon said combining, the liquid formulation isready for injection.
 16. The method of claim 15, wherein said combiningcomprises mechanical agitation.
 17. The method of claim 16, wherein saidcombining is performed in one minute or less.
 18. The method of claim15, wherein the dry formulation consists essentially of: dantrolenesodium consisting essentially of dantrolene sodium particles less thanabout 2 microns in average diameter; a water-soluble polysorbate; and acompound selected from the group consisting of sorbitol and mannitol.19. The method of claim 15, wherein the dry formulation furthercomprises polyvinylpyrrolidone (PVP).
 20. A method for treating anon-normothermia condition in mammal, comprising the step of:administering to a mammal in need of treatment for a non-normothermiccondition a therapeutically effective amount of a safe for injection,low volume liquid formulation of dantrolene sodium comprising:dantrolene sodium at a concentration in the range of 3.33 mg/ml to166.67 mg/ml; a water-soluble polysorbate; a compound selected from thegroup consisting of sorbitol and mannitol; and water as a liquidcarrier, wherein said dantrolene sodium and water are present togetheras a colloidal dispersion of dantrolene sodium particles in the water,wherein the dantrolene sodium particles are less than about 2 microns inaverage diameter, and wherein the formulation is safe for intravenousadministration.